Scoping review of cytolytic vaginosis literature

Background Cytolytic vaginosis (CV) is a little-known, controversial condition that is typically not considered for women presenting with vulvovaginitis symptoms. Objective: The objective of this scoping review was to identify and compile the global evidence on CV. Methods A medical librarian searched Prospero, Wiley Cochrane Library, Ovid Embase, Ovid Medline, EBSCO CINAHL, ProQuest Dissertations and Theses Global, and Scopus, from inception to April 4, 2019 and updated to October 17, 2021. Studies were eligible if they discussed CV. Two independent reviewers conducted study selection and data extraction. Results Sixty-four studies were identified, with 67% of studies (n = 43) published since 2007. Studies were from around the world, including the United States (28%, n = 18), Brazil (11%, n = 7), Portugal (11%, n = 7), and China (11%, n = 7). Fifty percent of studies (n = 32) were reviews; the remainder were observational; and of these, 78% (n = 25) were cross-sectional. The most frequent topics included: diagnosis (19%, n = 12), prevalence (17%, n = 11), and overview of CV (50%, n = 32). Evidence for prevalence in symptomatic women (median prevalence of 5%, interquartile range 3%-8%) was based only on 16% of studies (n = 10) with minimal evidence on prevalence in asymptomatic women and across different geographic regions. Microbiological findings, including abundant lactobacilli and fragmented epithelial cells, were found useful to distinguish between CV and vulvovaginal candidiasis, and Lactobacillus crispatus was noted to dominate the vaginal flora in women with CV. Most studies used subjective criteria to diagnose CV as the condition lacks gold-standard microscopic criteria. The suggested primary treatment (baking soda irrigations) was largely based on expert opinion, and there was minimal evidence on associations between CV and other conditions. Conclusion Knowledge gaps currently exist in all realms of CV research. Additional research is needed to confirm the validity of CV and ensure that women are diagnosed and treated effectively.


Introduction
Vaginitis has a high global prevalence and economic burden, and has a significant impact on woman's physical health, mental health, and overall function [1][2][3][4][5]. The differential diagnosis for vaginitis is broad (S1 Table) and it remains controversial whether a dysbiosis pattern seen on wet mount called cytolytic vaginosis (CV) should be included in the differential diagnosis.
Evidence about CV appears in literature as early as 1961 [6], yet, it was not until 1991 that Leonard Cibley and Laurence Cibley coined the term CV after encountering women with symptoms similar to vulvovaginal candidiasis (white discharge, irritation, pruritus) but with a markedly different pathophysiology and treatment [7]. They published a narrative paper on CV; it described this entity, proposed diagnostic criteria, and described treatment. However, the paper did not provide any quantitative patient data including demographics, symptoms, diagnosis results, and treatment outcome. Since then, CV has remained a largely unknown, controversial, and understudied condition. It is still questioned whether it is an actual condition, with some asserting that the symptoms are physiological [8], and it is typically not listed as a condition in vaginitis guidelines [9].
A critical appraisal of CV was published in 2020 and examined whether CV should be seen as a true condition [10]. Appropriate to a critical appraisal, the authors examined evidence from published articles they were aware of (n = 10) and provided an opinion on the existence of CV. However, there has not yet been a scoping review of CV. A scoping review is different from both a critical appraisal and a systematic review; instead of answering a specific question, it seeks to delineate evidence available, identify knowledge gaps, define concepts, or examine research methodology [11].
Our objective was to complete a scoping review of CV to uncover all evidence and identify knowledge gaps related to the following aspects of CV: prevalence, diagnosis, treatment, and associations between CV and other conditions.

Methods
We used the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines for scoping reviews (S2 Table) for reporting this review [12]. A review protocol for this study does not exist.

Search strategy
A medical librarian (S.C.) searched Prospero, Wiley Cochrane Library, Ovid Embase, Ovid Medline, EBSCO CINAHL, ProQuest Dissertations and Theses Global, and Scopus. All databases were searched from inception to April 4, 2019 and updated to October 17, 2021. The search strategy included both text words and controlled vocabulary (e. g., MeSH, EMTREE) for the concepts "Lactobacillus," "cytolysis," and "vaginal." No limits were applied. Results (219 studies) were exported to COVIDENCE citation management software, where duplicates (116 studies) were identified and removed. Search strategies and the PRISMA-S checklist are included in S1 File and S3 Table, respectively.

Study selection
Studies were included if they discussed CV. Studies did not need to use the term "cytolytic vaginosis," but they needed to indicate that the condition involved excess lactobacilli and cytolysis. Studies also needed to have a discussion on CV, however brief, to be included.
Two independent reviewers (R.K. and F.D.C.) first screened the abstract and the title of studies for eligibility and then reviewed the full text of the eligible studies to determine whether they met the inclusion criteria. References of the included studies that focused on CV were reviewed. Studies in other languages were translated into English with either Google Translate or an online translating service. Excel spreadsheets were used to track the selection of studies. Any disagreements were resolved with discussion, and we calculated the percentage of agreement between study selectors.

Data extraction
The data extracted from all relevant studies included: Descriptive data: (1) year published; (2) form of publication: journal, abstract, presentation, book, and chapter/section of book; (3) study location (if the study did not provide the location, the location of the authors was used as the study location); (4) language; (5) funding source; (6) journal title; (7) journal impact factor (2018 InCites Journal Citation Report); (8) type of study: review, case report, case series, cross-sectional descriptive, cross-sectional analytical, case control, prospective cohort, retrospective cohort, and randomized controlled trial; (9) CV focus of study: yes or no; (10) aspect of CV: prevalence, diagnosis, treatment, associations, comprehensive; (11) number of women; and (12) study objective.
Prevalence data: (1) participant selection: location of recruitment, date of recruitment, exclusion criteria, and sampling method; (2) participant age; (3) vulvovaginal symptoms: yes or did not indicate; (4) negative yeast microscopy; (5) negative yeast culture; and (6) number of women with CV (in total and in a subgroup).
Association: (1) exposure/outcome; (2) selection bias based on National Institutes of Health (NIH) quality assessment tools [13]; (3) information bias based on NIH quality assessment tools [13]; (4) confounder bias based on NIH quality assessment tools [13]; and (5) study results. Bias was assessed for studies with a focus on complication to help determine the credibility of the results. Data were extracted by at least two independent reviewers, including R.K., F.D.C., R.G., A. R., and O.B. Reviewers first calibrated data on the first few studies together and then extracted data independently. Disagreements were resolved through discussion, and we calculated the percentage agreement between data extractors.

Synthesis of results
The descriptive data, diagnosis data, and treatment data were analyzed in Excel and summarized in figures organized by attribute. The median prevalence was calculated by subgroup and plotted using a forest plot. Studies specific to diagnosis were organized by focus and shown in a table. Studies on associations between CV and other conditions were organized by type and listed in a table. The data synthesis was done in Word and Excel.

Study selection and data extraction
The total number of unique studies found was 524; of these, 64 met the selection criteria (43 from the original search, 16 from the reference search, and 5 from other sources Fig 1). The percentage of agreement between reviewers was 84% on study selection and 86% on data extraction. Table 1 lists the characteristics of the included studies, and the full data set is included in S4 Table. Descriptive statistics Table 2 provides a summary of the descriptive statistics.
The articles were published in a broad array of journals, including obstetrics/gynecology (58%, n = 33); general medical journals (7%, n = 4); and nurse practitioner journals (7%, n = 4). Thirty-seven percent of articles (n = 21) were published in journals without an impact factor, and of the journals with an impact factor, the median impact factor was 1.5.

Prevalence
Twenty-three of the 32 non-review studies (72%) provided sufficient information to enable us to calculate the prevalence of CV for the study; Fig 2 shows the median prevalence by subgroup.
Cross-sectional descriptive studies had a lower median prevalence compared to the other study types. The median prevalence values in the Czech Republic, India, and Iran were outliers, likely because their prevalence was only based on one study and the study focused on a subpopulation. Studies using wet mount to diagnose CV had the highest median prevalence and the widest range, while studies using the Pap test had the lowest median prevalence. The studies using the Pap test did not indicate whether the conventional Pap approach or liquidbased cytology (less sensitive for flora evaluation) was used, although two of these studies were done after liquid-based cytology became available. In addition, performing a Candida culture appeared to have little impact on reported prevalence, and pregnant women and women with recurrent symptoms had a higher median prevalence.

Diagnosis
Twenty-two of the 32 non-review studies (69%) provided diagnostic criteria. Table 3 summarizes the diagnostic criteria used in the studies. Nine of these studies (41%) focused primarily on microbiology results for diagnosis (lactobacilli and cytolysis), while the other 12 studies (55%) used Cibley criteria or a variant of Cibley criteria, and 1 study (5%) did not indicate. Two studies (9%) provided scores/observations for diagnosing CV. Specifically, Hu et al. [39] was based on quantity of lactobacilli and cytolysis; Hacisalihoglu et al. [36] evaluated lactobacilli, neutrophils, cytolysis, Candida ssp. hyphae, and Trichomonas vaginalis on a scale of 0-3, based on number of colonies per oil immersion field. The remainder of the studies used subjective terms to characterize lactobacilli and cytolysis; for instance, "massive," "abundant," and "increased" for lactobacilli and "crowded with cellular debris," "free nuclei," and "marked" for cytolysis.
Sanches 2020 [57] and Shopova 2006 [60] used the Nugent score (gold standard for bacterial vaginosis); as a component of their diagnosis, both studies reported CV samples had a Nugent score of nil. Five studies classified vaginal flora into grades, and only Moghaddam et al.'s study [47] included a grade specific for CV with both lactobacilli and cytolysis.
In addition, there were 12 (19%) studies specifically focused on the topic of diagnosis ( Table 4). Five of these studies examined how to distinguish between vulvovaginal candidiasis and CV and found that microbiological features appear to be more effective than clinical features. Four studies (6%) focused on Lactobacillus species and found that Lactobacillus crispatus dominates in women with CV; women with CV have less diverse lactobacilli microbiome; and Lactobacillus crispatus in women with CV secretes more acid. Two studies focused on wet mount findings for vaginal flora; the earlier study divided vaginal flora into grades based on lactobacilli and described CV as a variant of grade 1 (normal flora); and the later study

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Scoping review of cytolytic vaginosis reported that >95% of dyspareunia, discharge, and severe discomfort symptoms improved after a 10-day course of baking soda sitz baths [36]; and Lapina et al. treated women post cystocele surgery with sertaconazol vaginal suppository and an alkaline (pH 8-9) vaginal moisturizer and found that women with CV had 100% resolution of their symptoms [43]. Only Lapina et al. did further testing after symptomatic resolution, using pH, and found that pH normalized in women after the treatment [43].
We also looked at treatment recommendations among all studies due to the limited information from non-review studies. This is summarized in Table 5. Thirty-five (55%) studies mentioned treatment for CV, and 25 of these studies were review studies. The primary treatment recommended was baking soda; reasons centered around increasing the vaginal pH and decreasing the quantity of lactobacilli. Six of the 35 studies (17%) suggested discontinuing tampon use with the rationale that the menstrual flow is basic, raises the vaginal pH, and in doing so inhibits the growth of lactobacilli. Five of the 35 studies (14%) mentioned treating with antibiotics including amoxicillin/clavulanic acid or doxycycline if unable to take penicillin and a short course of vaginal 2% clindamycin cream or metronidazole. Several studies provided more general vaginitis treatment suggestions including discontinuing antifungal treatment, using only water and not soap to wash, and refraining from sexual intercourse until symptoms resolve. Table 6 shows the studies that examined conditions associated with CV with an assessment of bias. The details of the bias assessment are provided in S6 Table. There were 8 studies [13%] reporting associations between CV and other conditions that can be divided into 3 topics: pregnancy, cervical dysplasia, and other. Three studies looked at pregnancy and each focused on a different aspect: infertility [15], fetal impairment [74], and group B strep [55]. CV was found to be associated with an increased risk of infertility and an increased risk of fetal impairment, but both studies had a moderateto-high risk of bias [15,74]. The study on group B strep found that women with group B strep had increased odds of also having CV and had a low-to-moderate risk of bias [55].

Associations between CV and other conditions
Three studies examined whether there was any association between cervical dysplasia and CV: two studies found no association (low-to-moderate risk of bias) [61,65], while one study found CV was associated with a lower risk of developing a high-grade intraepithelial lesion or invasive neoplasia (moderate risk of bias) [49]. The remaining two studies found greater odds of CV in women with vulvodynia (low-to-moderate risk of bias) [64] and lower odds of their having vaginal candidiasis (high risk of bias) [47].  [7] on CV, and the cut-off for the subsequent groups was chosen to divide the remaining years equally between groups. https://doi.org/10.1371/journal.pone.0280954.g002

Discussion
This scoping review is the first systematic review to map out the literature published on CV. We uncovered more studies (64 vs. 10) than the 2020 critical appraisal [10] because we conducted a systematic literature search and our inclusion criteria included studies in foreign languages, and studies not focused on CV. Nevertheless, our scoping review had findings similar to the 2020 critical appraisal, including the need for objective criteria and the need for evidence on treatment and treatment outcomes.  Statistically significant clinical differences between CV and vulvovaginal candidiasis including 1) less swelling, erosions, and ulcerations in women with CV; 2) increased quantity of discharge and discharge described as more paste-like in women with CV; and 3) symptoms primarily during ovulation and the luteal phase of the menstrual cycle with CV compared to being more evenly spread out with vulvovaginal candidiasis. We calculated likelihood ratios (S5 Table), and none of the 3 symptoms/signs are individually useful for diagnosis.

Microbial composition
Fontan 2020 [31] Spain 38 Whether Lactobacillus crispatus can be used as a marker for CV L. crispatus prevalence in the CV group was 73.3% and 16.6% in the "normal microbiota" group. Use high-throughput sequencing to identify biomarkers for CV 1) There was increased microbial diversity in the normal flora group; 2) The density of Lactobacillus colonies was higher in CV group compared to normal flora group; 3) In CV, L. crispatus made up 97.5% of Lactobacillus species compared to 40% in the normal flora group.
Yang 2020 [73] China 149 Microbial composition and variation in Lactobacillus microbiome in patients with CV compared to healthy controls 1) CV group had a less diverse Lactobacillus species; 2) L. crispatus had a higher prevalence in the CV group than the healthy flora group (88.7 vs. 56.4%); and 3) L. crispatus from the CV group produced more acid than L. crispatus from the healthy flora group.

Other
Azevedo 2019 [19] Portugal 50 Impact of sampling site on wet mount microscopy results In CV, there was a higher sensitivity rate for anterior fornix samples; however, this was not statistically significant. Why there is a paucity of studies on CV compared to the over 7000 studies that have been published on vaginitis [10] is unclear; is it because CV is unknown to the medical community or because it is a variant of the normal vaginal microbiome? However, given that studies of CV span 3 continents, are from diverse countries, and are published in a broad spectrum of journals, it is more suggestive that CV is a true condition.
CV is not the only vaginal microbiome dysbiosis condition that is little known and understudied. Aerobic vaginitis or desquamative inflammatory vaginitis is similar to bacterial vaginosis in that it lacks lactobacilli, but dissimilar in that the vaginal microbiome is colonized predominately by aerobic bacteria rather than by anaerobic bacteria [75]. The symptoms of aerobic vaginitis include excessive vaginal discharge, pruritis, burning, and dyspareunia [75]. In addition, there is also a controversial entity characterized by the presence of abnormally long possible lactobacilli (length of 40 um-75 um instead of 5 um-15 um), referred to as leptothrix, fusiform lactobacilli, and lactobacillosis; it is found to coexist with other vaginal  dysbiosis and infectious conditions as well as normal flora [66,76]. It often does not have symptoms (based on author experiences), unclear whether it is causative or incidental [77], and unknown whether it is an abnormally long Lactobacillus sp. or a different bacteria species [76][77][78].
Although the studies in this scoping review can be used to provide an estimate of the median prevalence of CV (5% [IQR 3%-8%] in women with symptoms and 22% [IQR 19%-24%] in women with recurrent symptoms), and the differences in prevalence among subgroups help provide credibility to the prevalence estimate, this estimate is limited by 1) quality of studies; 2) lack of standard criteria used to diagnose CV; and 3) insufficient number of studies overall and in subgroups. To inform clinicians whether and how much CV should be considered, further studies on prevalence using gold-standard diagnostic criteria in symptomatic women and asymptomatic women in various geographic locations are needed.
The subjective criteria used by studies in this scoping review to diagnose CV highlight the need to have a gold-standard objective criterion. There is some movement in this direction; for instance, Hu et al. found distinct differences in quantity of Lactobacillus spp., percentage of fragmented epithelial cells, and percentage of whole epithelial cells between women presenting with CV and other vaginosis conditions [39], whereas Hacisalihoglu et al. scored cytolysis, lactobacilli quantity, neutrophils, finding of bacterial vaginosis, Candida spp. hyphae/spores, and Trichomonas vaginalis based on quantity under oil immersion (0-3 scale) [36]. However, they reported only individual scores rather than also providing a composite score.
Perhaps summarizing the percentage of cytolysis, quantity of lactobacilli, and lack of other vaginosis findings into a composite score similar to Donder's criteria for aerobic vaginitis [75] or Nugent's score for bacterial vaginosis [79] may be a way forward. This should be possible using either a wet mount or a Gram stain. The wet mount is advantageous as it enables clinicians to diagnose CV quickly during a clinic visit; however, in practice, Gram stains are usually completed at the laboratory due to the lack of microscopes and expertise in clinic [68,80]. Another option may be modeling it after Hay/Ison criteria, which classify vaginal flora into grades and require less skill and time [81]. In addition, with the possible shift to molecular diagnosis including a nucleic acid amplification test [67], a gold standard may need to take this into account as well. Fig 3 shows illustrations of wet mounts and gram stains for normal, bacterial vaginosis, and CV.
The vaginal community state type is the framework often used to categorize the vaginal microbiome [82]. In our scoping review, no studies that matched the inclusion criteria explicitly examined how CV fits into this framework. However, there were studies that examined the microbiology of CV and found the Lactobacillus crispatus dominates which is most consistent with vaginal community state type I [83].
There were only a few studies evaluating treatment for CV, and the results infer that increasing vaginal pH with baking soda is effective. However, these studies were observational, primarily included a single exposure and outcome, had a small number of participants, and did not include microbiological results post-treatment. Studies with a more rigorous design, including randomized controlled trials, would be useful to further delineate treatment effectiveness. In addition, it would be advantageous for studies to explore more definitive treatment options. Other vaginal dysbiosis conditions (for instance, bacterial vaginosis) are treated with antibiotics or antiseptics with a curative intent [66].
Some studies examined associations between CV and other conditions such as cervical dysplasia and pregnancy/fertility. However, these studies have, on average, a moderate risk of bias and there are few such studies, so it is difficult to make any inferences. There was an additional prospective cohort study on pregnancy outcomes of 2453 women by Bercovici et al. [84] in 1973 that found cytolysis increased from first to second to third trimester before decreasing prior to delivery; the incidence of cytolysis was significantly higher in women with hyperemesis gravidarum and diabetes and did not appear to have any adverse fetal outcomes. However, the study did not consider the quantity of lactobacilli [84].
It is possible that we missed capturing studies on CV as we did not examine gray literature and only reviewed citations of studies that focused on CV. However, given that our scoping review included more studies than previous reviews, it is unlikely that any potentially missed

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studies would significantly impact our results. Due to limited resources, we only assessed bias of studies that focused on conditions associated with CV, as it was most important to determine bias for these studies. Our review of treatment included primary and secondary sources and as such, it is possible that some information was repetitive; however, secondary sources were included because it was difficult to discern whether authors' experiences with treatment were included in studies that referenced treatment recommendations.

Conclusion
This scoping review clearly shows that there is a lack of robust evidence along all aspects of CV. Historically, CV has been discounted based on lack of evidence, and its symptoms have been explained as simply physiological or even psychological. However, we feel that it is important to consider CV, given the volume of consistent evidence supporting this condition from a diverse range of countries and sources, and the potential for distressing symptoms if left untreated. Future research should especially be centered around establishing gold-standard diagnostic criteria that will enable practitioners, laboratories, and researchers to better characterize, diagnose, and confirm the validity of this equivocal condition.
Supporting information S1  Table. Bias assessment of studies on the association between cytolytic vaginosis and other conditions. (PDF) S1 File. Search strategies. (PDF)